Method for detecting inflammation and inflammatory conditions

ABSTRACT

Methods for detecting the inflammatory biomarkers molecule CD 163 in biological samples are provided. Also provided are methods for monitoring the course of an inflammatory process or condition in a patient and compositions and methods for preventing and treating inflammation and inflammatory processes.

INTRODUCTION

[0001] This invention was supported in part by funds from the U.S.government (NIH Grant No. AI40686) and the U.S. government may thereforehave certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Mononuclear phagocytes (monocytes and macrophages) are criticalcomponents of both innate and acquired immunity and are found invirtually every tissue of the body, including the central nervoussystem. Mononuclear phagocytes participate in both antibody dependentand independent cytotoxicity, phagocytosis and killing of bacteria,destruction of effete erythrocytes, presentation of antigens for T cellactivation, and secretion of a wide variety of inflammatory cytokines.

[0003] The secretion of inflammatory cytokines, as well as mononuclearphagocyte effector functions, are greatly influenced by solublemediators. For example, priming by interferon gamma (IFNγ) and exposureto lipopolysaccharide, tumor necrosis factor alpha (TNFα), interleukin-1(IL-1), or granulocyte-macrophage colony stimulating factor (GM-CSF) canstimulate mononuclear phagocytes to secrete inflammatory cytokines suchas TNFα, IL-1 and interleukin-6 (IL-6) (Auger, M. J. and J. A. Ross.1992. In: The Macrophage: the natural Immune System, New York: OxfordUniversity Press, pp. 1-74). Interleukin-10 (IL-10; originally known ascytokine synthesis inhibitory factor) has been shown to inhibit theexpression of a wide range of inflammatory cytokines in vitro (Berkman,N. et al. 1995. J. Immunol. 155:4412-4418; de Waal Malefyt, R. et al.1991. J. Exp. Med. 174:1209-1220) as well as in vivo (Chernoff, A. E. etal. 1995. J. Immunol. 154:5492-5499; van der Poll, T. et al. 1997. J.Immunol. 158:1971-1975). Glucocorticoids, interleukin-4 (IL-4) andinterleukin-13 (IL13) have also been shown to down regulate theexpression of inflammatory cytokines produced by mononuclear phagocytes.In addition to inhibiting the release of inflammatory cytokines,glucocorticoids have also been shown to upregulate the expression ofCD163 on mononuclear phagocytes (Hogger, P. et al. 1998. Pharm. Res.15:296-302; Hogger, P. et al. 1998. J. Immunol. 161:1883-1890; Wenzel,I. et al. 1996. Eur. J. Immunol. 26:2758-2763).

[0004] CD163 is a mononuclear phagocyte restricted antigen which is amember of the cysteine rich scavenger receptor family group B. Normalhuman macrophages stain brightly for CD163 and glucocorticoid treatmentin vivo increases CD163 expression (Zwadlo-Klarwasser, G. et al. 1992.Int. Arch. Allergy Immunol. 97:178-180; Zwadlo-Klarwasser, G. et al.1990. Int. Arch. Allergy Immunol. 91:175-180). It has been suggestedthat these CD163 bright macrophages may play a role in the resolution ofinflammation as they are found in high numbers in inflamed tissues(Zwadlo, G. et al. 1987. Exp. Cell Biol. 55:295-304) and have been shownto release an incompletely characterized anti-inflammatory mediator(Zwadlo-Klarwasser, G. et al. 1995. Int. Arch. Allergy Immunol.107:430-431).

[0005] One mononuclear phagocyte marker that bears a strikingresemblance to CD163 is p155 (Morganelli, P. et al. 1988. J. Immunol.140:2296-2304). Expression of this 134 kDa (non-reduced)/155 kDa(reduced) glycoprotein is restricted to mononuclear phagocytes andupregulated by glucocorticoid treatment. It has now been found thatCD163 is identical to P155 and that this molecule could have activity asan anti-inflammatory molecule. Thus, this glycoprotein is believed to beuseful as a biomarker for inflammation and inflammatory conditions andprocesses in humans.

[0006] A method has now been developed for detection of CD163 in humanplasma. This method is useful in monitoring inflammation andinflammatory processes in humans.

SUMMARY OF THE INVENTION

[0007] An object of the present invention is to provide a method fordetecting CD163 in a biological sample, preferably a plasma sample,which comprises contacting the biological sample with a CD163 captureantibody and a CD163 detection antibody, so that levels of CD163 in thebiological sample can be quantified. The method of the present inventionis particularly useful for monitoring the course of an inflammatorycondition or process.

[0008] Another object of the present invention is to provide acomposition for prevention and treatment of inflammation which comprisesCD163. In one embodiment, the composition further comprises aglucocorticoid.

[0009] Another object of the present invention is to provide a methodfor reducing signs and symptoms of inflammation which comprisescontacting cells or tissues with the CD163 molecule, either alone or incombination with a glucocorticoid.

[0010] Yet another object of the present invention is to provide amethod for preventing or treating inflammation in an animal whichcomprises administering to an animal an effective amount of acomposition comprising CD163 either alone or in combination with aglucocorticoid.

DETAILED DESCRIPTION OF THE INVENTION

[0011] CD163 is a glucocorticoid inducible member of the scavengerreceptor cysteine rich family of proteins. It is known that CD163 ishighly expressed on human macrophages but has been reported to be foundon less than 50% of peripheral blood monocytes. It has now been foundthat, contrary to previous reports, more than 99% of all CD14 positivemonocytes express CD163. It has also been found that IL-10, likeglucocorticoids, induces higher CD163 expression on cultured humanmonocytes. Glucocorticoid-induced CD163 expression has also beenexamined and found to be due to an IL-10 independent mechanism since itwas not inhibited by anti-IL-10 and was additive with IL-10 treatment.It has also been found that p155, a previously identifiedmonocyte/macrophage marker of unknown function, is the same as CD163.The fact that CD163 is upregulated by potent anti-inflammatory mediatorssuch as glucocorticoids and IL-10 indicates that CD163 may be animportant anti-inflammatory molecule and a potential biomarker forinflammation and inflammatory conditions.

[0012] Previous studies using mAbs RM3/1, Ber-Mac3 and others hadreported that only 0%-40% of circulating monocytes are positive forCD163 (Hogger, P. et al. 1998. Pharm. Res. 15:296-302; Hogger et al.1998. J. Immunol. 161:1883-1890; Zwadlo, G. et al. 1987. Exp. Cell Biol.55:295-304; Backe, E. et al. 1991. J. Clin. Path. 44:946-953; van denHeuvel, M. et al. 1999. J. Leuk. Biol. 66:858-866). However, previousstudies with another antibody to p155, a molecule that has been shown tobe identical to CD163, Mac 2-48, has consistently demonstrated thatvirtually all freshly isolated monocytes are positive for CD163. Toaddress the possibility that sub-optimal detection of the lower affinityRM3/1 and Ber-Mac3 antibodies (previously used only with FITC labeledsecondary antibodies) might account for this discrepancy, freshlyisolated PBMCs were stained with FITC conjugated AML 2.23 (anti-CD14)and biotinylated RM3/1 or biotinylated Mac248, followed by detectionwith SAPE.

[0013] Although Mac2-48 staining was slightly higher, virtually all CD14bright PBMCs were positive for both RM3/1 and Mac 2-48, while most CD14dim or negative PBMCs were negative. When PBMCs were gated for CD14bright cells, greater than 99% were positive for both RM3/1 and Mac2-48, while the P3 control mAb detected less than 1% of the gated cells.Virtually identical results are obtained when highly purified monocyteswere used in place of fresh PBMCS. These results indicate that CD163 isexpressed on nearly all CD14-positive circulating monocytes.

[0014] To assess whether different cytokines could influence CD163expression, freshly isolated PBMCs were cultured for 24 hours in thepresence of various cytokines, 200 nM DEX (as a positive control forCD163 upregulation), or control media. The cells were then subjected tostaining and flow cytometric analysis.

[0015] Treatment of PBMCs for 24 hours with IL-10 alone or the syntheticglucocorticoid DEX alone increased monocyte CD163 expression byapproximately 4- and 7-fold, respectively (p<0.01). Combined DEX plusIL-10 treatment resulted in significantly higher CD163 expression thanwhen monocytes were cultured with DEX only or IL-10 only, indicating anadditive effect when glucocorticoid treatment was used in conjunctionwith IL-10 (p<0.01). None of the other cytokines tested had astatistically significant effect on monocyte CD163 expression at theconcentrations used, and none significantly increased or decreased theDEX-induced upregulation of expression. The increased expression ofCD163 by IL-10 and glucocorticoid treatment was also demonstrated bywestern blots of monocyte lysates. These data indicated that CD163upregulation plays an important role in the anti-inflammatory actions ofglucocorticoids.

[0016] In order to determine whether the increased expression of CD163on monocytes is due to increased RNA and protein synthesis, northernblots were performed on monocyte lysates. Monocytes were treated for 8hours with either IL-10, the glucocorticoid FP or control media. CD163mRNA levels increased from undetectable to strong bands with theaddition of IL-10 or FP. This indicated that the induction of CD163 byIL-10 or glucocorticoids is due, at least in part, to increased RNA andprotein synthesis.

[0017] A dose-response relationship between IL-10 treatment and CD163expression was established by culturing PBMCs for 24 hours in thepresence of 0.1 to 100 ng/ml IL-10. The results were a sigmoidal doseresponse curve when levels of CD163 expression were related to IL-10concentration. CD163 expression was increased approximately 3.5 fold by10 and 100 ng/ml IL-10 treatment when compared to control (p<0.01).

[0018] It was possible that, in addition to direct effects onmononuclear phagocytes, DEX might upregulate CD163 expression indirectlyby altering the amount of IL-10 produced by lymphocytes. In order totest this possibility, PBMCs were cultured with IL-10, DEX or controlmedia in the presence or absence of a blocking anti-IL-10 IgG. Resultsshowed that expression of CD163 was not significantly affected by thepresence of anti-IL-10 in control or DEX treated cells. However, IL-10upregulation of CD163 expression was inhibited by anti-IL-10 (p<0.01),where CD163 expression was reduced to near control levels. Thesefindings indicate that DEX increased CD163 expression by an IL-10independent mechanism.

[0019] In addition to testing the effects of cytokines in combinationwith glucocorticoids on levels of CD163, studies were performed toexamine the effects of glucocorticoids in combination withlipopolysaccharide (LPS). Monocytes cultured with LPS alone had lowlevels of CD163 detected on their surface using an immunofluorescencetechnique for CD163 detection. Treatment of cells with DEX or IL-10alone increased expression of CD163, as had been previously shown.However, when monocytes were cultured for 48 hours with DEX combinedwith LPS, their was a synergistic increase in CD163 expression, wherethe effect of DEX alone was increased by more than 2-fold when LPStreatment was added.

[0020] Additional in vitro studies with LPS showed that LPS inducesshedding of monocyte surface CD163 within 2 hours, a result that wasconsistent with studies by others using the phorbol ester PMA. PMA hasbeen shown to induce rapid shedding of surface CD163 from monocytes inculture, an effect that was blocked by protease inhibitors (Droste, A.Et al. 1999. Biochem. Biophys. Res. Commun. 256:110-113). Therefore,like PMA, LPS is capable of inducing CD163 shedding. LPS-inducedshedding occurred even with monocytes that had been cultured for 48hours in DEX and thus had 5- to 10-fold higher levels of surface CD163than freshly isolated monocytes. In cells that had increased levels ofCD163 due to treatment with glucocorticoids plus LPS, as reported above,the surface CD163 molecules are largely resistant to shedding induced bysubsequent treatment with LPS, although they remained sensitive toPMA-induced shedding. This LPS-conferred resistance to subsequentLPS-induced shedding of CD163 is similar to reported endotoxinpre-conditioning for resistance to subsequent inflammatory insults.

[0021] The effect of the cytokine IL-10 was shown to be unique among thecytokines tested in that, like glucocorticoids, it augmented CD163expression on freshly isolated mononuclear phagocytes. This increase inCD163 is thought to be a direct effect on monocytes, as studies usinghighly purified monocytes or the established human monocyte cell lineTHP-1 yielded results that were in agreement with those performed usingPBMCs. In contrast, a number of other cytokines (including IL-4 andIL-13) did not upregulate CD163 expression at the concentrations tested.Even though IL-4, IL-10 and IL-13 have all been reported to inhibitmonocyte production of inflammatory cytokines such as TNFA (Cosentino,G. et al. 1995. J. Immunol. 155:3145-3151; Joyce, D. A. et al. 1996.Cytokine 8:49-57; Joyce, D. A. et al. 1996. J. Interferon Cytokine Res.16:511-517), differential regulation of mononuclear phagocyte surfacemolecules by IL-10 and IL-4/IL-13 is not without precedent. For example,CD64, like CD163, is upregulated by IL-10, but not by IL-4 or IL-13 (deWaal Malefyt, R. et al. 1993. J. Immunol. 151:6370-6381; te Velde, A.A.et al. 1992. J. Immunol. 149:4048-4052).

[0022] When given in combination with DEX, IL-10 is the only cytokinetested that significantly increased CD163 expression over DEX treatmentalone. Since the concentration of DEX used is >90% saturating for theglucocorticoid receptor and the dose of IL-10 used gives maximal CD163induction, the additive effect of these treatments suggests thatglucocorticoids and IL-10 influence CD163 expression by independentmechanisms. This conclusion is further supported by the finding that ananti-IL-10 antibody (which blocks the biological activity of IL-10)reduced the IL-10 induction of CD163 to control levels, but had noeffect on the DEX induction of CD163. This demonstrates that theglucocorticoid effect is not dependent on elevated levels ofextracellular IL-10 and does not upregulate CD163 expression by firstincreasing IL-10 synthesis and release.

[0023] The finding that either GM-CSF or IL-4 plus DEX does not enhanceCD163 expression over DEX treatment alone contrasts with that of arecent report. While Van den Heuvel and colleagues (van den Heuvel, M.et al. 1999. J. Leuk. Biol. 66:858-866) found that neither GM-CSF norIL-4 alone had any effect on CD163 expression, they detected asynergistic effect using either GM-CSF or IL-4 plus DEX. This disparityis likely due to differences in experimental procedures such asisolation technique, culture conditions and duration of stimulus. In theprevious report, monocytes were purified by gradient centrifugation,lymphocyte resetting and monocyte adherence while the present studiesused density centrifugation purified PBMCs. Furthermore, cells weretreated for 24 hours, while in the previous study cells were treated for2 days.

[0024] The dose response curve for the IL-10 effect on CD163 expressiondemonstrates a dynamic range of IL-10 concentrations that is from 0.1ng/ml to 10 ng/ml. This is consistent with previous findings concerningthe effect of IL-10 on a wide range of monocyte functions such astissue. factor expression and associated procoagulant activity(Ernofsson, M. et al. 1996. Br. J. Haematol. 95:249-257; Ones, L. T. etal. 1996. Cytokine 8:822-827), as well as MIP-1α (Berkman, N. et al.1995. J. Immunol. 155:4412-4418), metallo-proteinase (Lacraz, S. et al.1992. J. Clin. Invest. 90:382-388) and TNF receptor (Hart, P. H. et al.1996. J. Immunol. 157:3672-3680) expression.

[0025] The fact that CD163 is upregulated by potent anti-inflammatorymediators such as glucocorticoids and IL-10 indicates that this CD163may be an important anti-inflammatory molecule. Further, these dataprovide support for the use of CD163 detection in biological samples,such as blood or plasma, as a means for detecting the presence ofinflammation or inflammatory conditions in patients.

[0026] In order to provide for use of CD163 as a biomarker ofinflammation, a method for detection of CD163 in biological samples suchas plasma was developed. The assay of detection is an ELISA assay usinga CD163-specific antibody such as MAC2-158 or MAC2-48 as the CD163capture antibody and the commercially available biotinylated antibodyRM3/1 as the CD163 detection antibody. Briefly, plates were coated withpurified MAC2-158 or MAC2-48 antibody and incubated overnight at 4 C.After washing, non-specific binding was blocked by adding blockingbuffer to each plate well and incubating for 30 minutes at roomtemperature. After washing, plasma samples to be tested were added andthe plates are incubated overnight at 4 C. or at room temperature for 2hours. After washing, the detection antibody was added, RM3/1, and theplates agin incubated. A streptavidin alkaline phosphatase tag was usedand the plates were developed.

[0027] Using this assay, relative levels of CD163 were assayed in theplasma of 4 patients undergoing cardiac surgery performed withnormothermic cardiopulmonary bypass. It is known that cardiac surgicalpatients exhibit a reproducible acute, inflammatory response asindicated by a rise in TNF, IL-6 and cortisol, followed by hepaticrelease of acute phase proteins. This response may be caused by severalmechanisms, including tissue trauma, ischemia-reperfusion injury,exposure to foreign membranes (when cardiopulmonary bypass is used) andtransient endotoxemia. In 4 of 4 samples from these patients that weretested, plasma CD163 increased approximately twofold at 60 minutesfollowing cardiopulmonary bypass, and returning to slightly belowbaseline levels on post-operative day 1. In addition, levels of CD163 inplasma of these patients was shown to correlate with levels ofinterleukin-6 (IL-6) in plasma. This is an important finding becauseprior to surgical stress, infection, or other inflammatory processes,there is no detectable IL-6 in plasma of humans. Therefore, these datademonstrate the link of CD163 time-course with other markers ofinflammation and provide the first demonstration that soluble CD163 actsas an acute phase protein during an inflammatory response.

[0028] In order to more closely mimic the in vivo inflammatory responseto infection in a more controlled setting than the cardiac patientsdescribed above, healthy volunteers were administered a 4 ng/kg bolusinfusion of LPS and monitored the levels of soluble CD163 in plasma. Asdescribed above, LPS had been shown to induce shedding of CD163 frommonocytes in vitro. Plasma samples were taken at baseline (beforeadministration of LPS), and then at various time points after LPSinfusion up to 72 hours after infusion initiation. Levels of CD163 inplasma were measured using the assay of the present invention. SolubleCD163 levels in plasma increased as much as 7-fold compared to baselinelevels, peaked at 1 to 2 hours, and remained elevated in 4 of 5volunteers at 12 hours post LPS administration. In the one volunteerwhere levels had declined at 12 hours, levels remained elevated up to 4hours before returning to baseline levels by 8 hours. Changes in levelsof known acute phase proteins, glucocorticoids, and pro- andanti-inflammatory cytokines were also monitored in order to determine iftheir levels correlated with the appearance of soluble CD163 in plasma.Levels of CRP, an acute phase plasma protein secreted in hepatocytes inresponse to an inflammatory stimulus, increased in plasma by 8 hours butdid not peak until 24 hours post LPS administration. Plasma levels ofTNFA, IL-6 and IL-10, all known to be produced following LPS infusion,peaked at 1, 2, and 4 hours post LPS administration, respectively.Plasma cortisol levels began to increase at 4 hours post LPSadministration but did not peak until 6 hours. Theses data demonstratedthat soluble CD163 is one of the earliest changes induced by an acuteinflammatory response that can be detected in plasma. Therefore, CD163acts as an early signaling event in the inflammatory response cascade.

[0029] Accordingly, the present invention provides a method fordetecting levels of CD163 in biological samples from individuals knownto have or suspected of having inflammation or inflammatory conditions.By biological samples it is meant to include, but is not limited to,plasma, whole blood, serum, urine, sputum, semen, cerebrospinal fluid,or synovial fluid. The inflammatory condition can be due to variety ofcauses including, but not limited to, lupus, rheumatoid arthritis,infection, and surgery. The method involves contact of a biologicalsample, such as plasma, with a CD163-specific antibody such as MAC2-158or MAC2-48, and then detection in the ELISA assay with a biotinylatedantibody such as RM3/1. This method is particularly useful in monitoringfor the presence or course of inflammation or inflammatory conditions ina patient.

[0030] The present invention also relates to compositions comprisingCD163 for use in the prevention and treatment of inflammation inanimals, including humans. In one embodiment of the invention, cells ortissues are contacted with CD163 and inflammation is prevented,suppressed or reversed. In another embodiment, a composition comprisingCD163 in a pharmaceutically acceptable vehicle is administered to ananimal suffering from inflammation or an inflammatory disease so thatthe inflammation or inflammatory disease is treated. Successfultreatment is indicated by a reduction in the signs and symptoms ofinflammation including a reduction in the presence of inflammatorymediators, such as cytokines. CD163 can be administered alone or incombination with another anti-inflammatory agent such as aglucocorticoid. In the context of the present invention, “effectiveamount” is an amount of CD163 capable of producing a desiredpharmacological effect such as a reduction in the signs and symptoms ofinflammation. Selection of additional anti-inflammatory agents to beadministered in conjunction with CD163 can be performed routinely by oneof skill in the art. Selection of the amount of CD163 to be administeredcan also be performed routinely by one of skill based upon results suchas the cell culture studies presented herein.

[0031] The following non-limiting examples are provided to betterillustrate the present invention.

EXAMPLES Example 1 Isolation and Culture of Peripheral Blood MononuclearCells (PBMCs)

[0032] PBMCs were isolated from heparinized human whole venous bloodusing Ficoll-Hypaque (d=1.077g) after the method of Böyum (Boyum, A.1968. Scand. J. Clin. Lab. Invest. Suppl. 97:77-89) . PBMCS were thenwashed three times with hepes buffered RPMI 1640 (Hazelton Biologicals,Lenexa, Kans.)/0.05% gentamicin (Elkins-Sinn, Inc., Cherry Hill,N.J.)/1% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan,Utah).

[0033] For cytokine treatment studies, isolated PBMCs were suspended inhepes buffered RPMI 1640/0.05% gentamicin/10% FBS at a concentration of2.0×10⁶ to 2.5×10⁶ cells/ml and cultured in 96 well plates at 37° C. and5% CO₂ in the presence of various mediators (Table 1). Mononuclear cellswere stained for flow cytometric analysis after 24 hours in cultureunless otherwise indicated. This enhanced cell recovery becausemonocytes, which initially adhere to plastic vessels, transiently detachfrom culture wells at 24-48 hours.

Example 2 Staining and Flow Cytometric Analysis

[0034] All staining procedures were performed at 4 C. Briefly, culturedPBMCs were incubated with normal human IgG (6 mg/ml) to block Fcreceptor-specific binding of mAbs and 30 μg/ml of the isotype controlmAb P3 or a saturating amount of mAb MAC2-48 (20 μg/ml) for one hour.Cells were then washed and stained for one hour with 17.5 μg/ml FITClabeled goat F(ab=)₂ anti-mouse Ig. The cells were again washed andfixed with 1% methanol free formalin.

[0035] For two color studies, cells were stained for one hour with 20μg/ml biotinylated MAC2-48, RM3/1, or P3 plus 20 μg/ml FITC AML 2.23 orFITC control mouse mAb in the presence of at least 2 mg/ml normal humanIgG in a total volume of 60 ml. After staining with primary mAbs, cellswere washed and stained with SAPE at a 1:40 dilution. Flow cytometricanalysis was performed on washed, unfixed cells soon after staining.

[0036] Cell fluorescence of monocytes gated using forward and sidescatter was analyzed using a Becton Dickenson FACScan (Franklin Lakes,N.J.). Mean fluorescence intensity (MFI) was calculated by subtractingthe MFI of the P3 stained mononuclear cells from the MFI of thecorresponding Mac 2-48 stained cells.

Example 3 Northern Hybridization

[0037] Human monocytes were isolated and cultured overnight as describedfor western blots. Monocytes were then stimulated for 8 hours with 5ng/ml IL-10 (R&D Systems) or 10⁻⁸ M FP. Total RNA was isolated fromIL-10 stimulated, glucocorticoid stimulated and control monocytes asdescribed by Dreier, et al. (Dreier, J. et al. 1998. DNA Cell Biol.17:321-323). 10 μg of total RNA per sample were electrophoreticallyseparated in a 1% agarose, 2% formaldehyde gel and transferred onto aHybond N⁺ nylon membrane (Amersham Inc., Arlington Heights, Ill.) in 20×saline-sodium citrate (SSC) using an LKB 2016 VacuGene blottingapparatus. Antisense RNA probes for northern hybridization weregenerated from linearized DNA templates using a digoxigenin RNA labelingkit (Boehringer Mannheim, Mannheim, Germany) and T7 RNA polymerase (NewEngland Biolabs, Schwalbach, Germany) as described by the manufacturer.Prehybridization was performed at 68° C. for 1 hr in a high SDShybridization buffer (7% SDS, 5× SSC, 50% formamide, 50 mM sodiumphosphate, 2% casein, 0.1% N-lauroylsarcosine, pH 7.0). Subsequently theheat-denatured probes (10 minutes at 95° C.) were added to thepre-hybridization solution (100 ng/ml) and hybridized at 68° C. for 16hours. The nylon membrane was washed twice for 5 minutes at roomtemperature in a 2× SSC, 0.1% SDS solution and twice for 15 minutes at68° C. in 0.5× SSC and 0.1% SDS. The hybridization results werevisualized by chemiluminescent detection with anti-digoxigeninFab=fragments conjugated with alkaline phosphatase and substrate CSPD asdescribed by the manufacturer (Boehringer Mannheim). Equal loading ofsamples was examined by hybridization of RNA with an actin antisense RNAprobe.

Example 4 ELISA Assay for CD163

[0038] ELISA plates were coated, 100 μl per well, with 5 μg/ml purifiedMAC2-158 (coating buffer of 0.1 M NaHCO₃0.5 M NaCl, adjusted to pH 8.4with HCl). Plates were incubated overnight at 4 C. and then washed 4times with wash buffer (1× phosphate buffered saline and 0.05% Tween20). Nonspecific binding was blocked by adding 200 μl blocking buffer toeach well (phosphate buffered saline with 10% FBS) and incubating theplates for 30 minutes at room temperature. Plates were then washed 3times with wash buffer. 100 μl plasma (1:10 dilution) was added and theplates incubated overnight at 4 C., or for 2 hours at room temperature.Each plate was washed 4 times with wash buffer. The detection antibodyRM3/1 was added in blocking buffer (100 μl; phosphate bufferedsaline+10% FBS)) and the plates were incubated for 1 hour followed bywashing 4 times with wash buffer. Streptavidin alkaline phosphatase(1/1000) was added in blocking buffer and the plates were incubated for30 minutes at room temperature, followed by 4 washes with wash buffer.The reaction was developed with a PNPP system by dissolving one 15 mgPNPP tablet (Sigma Chemical Co.) in 15 ml PNPP diluent (0.05 M Na₂CO₃,0.001 M MgCl₂, pH 9.75) and adding 100 μl of solution to each well.Plates were developed for 5 to 30 minutes. 100 μl 1 M NaOH was added tostop the reaction. The plates were read on a spectrophotometer at 405nM.

What is claimed is:
 1. A method for detecting the presence of CD163 in abiological sample comprising: a) contacting the sample with a CD163capture antibody to form a CD163-antibody complex; and b) contacting theCD163-antibody complex with a CD163 detection antibody so that levels ofCD163 in the sample are detected.
 2. The method of claim 1 wherein theCD163 capture antibody is MAC2-158 or MAC2-48 and the CD163 detectionantibody is RM3/1.
 3. The method of claim 1 wherein the biologicalsample is human plasma.
 4. A method for monitoring the course of aninflammatory condition or inflammatory process comprising detecting thepresence of CD163 in biological samples via the method of claim
 1. 5. Acomposition for prevention or treatment of inflammation comprisingCD163.
 6. The composition of claim 5 further comprising aglucocorticoid.
 7. A method for reducing the signs and symptoms ofinflammation comprising contacting cells or tissues with the compositionof claim
 5. 8. A method for reducing the signs and symptoms ofinflammation comprising contacting cells or tissues with the compositionof claim
 6. 9. A method for preventing or treating inflammation in ananimal comprising administering to the animal an effective amount of thecomposition of claim
 5. 10. A method for preventing or treatinginflammation in an animal comprising administering to the animal aneffective amount of the composition of claim 6.